The present invention relates generally to amino-terminal-modified (N-terminal-modified) chemokines and the use of such chemokines to inhibit the interaction between chemokine receptors and naturally occurring ligands of those receptors. More specifically, the invention relates to the expression in host cells of recombinant polynucleotide sequences encoding chemokines having additional amino acids or other chemical groups attached to their amino termini, and the use of such N-terminal-modified chemokines as research tools for identifying chemokine receptors, as vaccine adjuvants, as agents for the chemotactic recruitment of migratory cells, as agents for the stimulation or inhibition of angiogenesis, as agents against autoimmune diseases and inflammation, and as agents to inhibit the binding of HIV to certain receptors and the infection of susceptible cells by HIV.
Chemokines (or chemotactic cytokines) are a class of cytokine molecules capable of chemotactically attracting migratory cells, and are involved in cell recruitment and activation in inflammation. Chemokines generally have small molecular weights in the range of 8-10 kDa and, like other small proteins such as cytokines, are believed to be rapidly inactivated in vivo, resulting in relatively short biological half-lives for these proteins. Most chemokines can be divided into two subgroups, CXC (alpha chemokines) or CC (beta chemokines), on the basis of the spacing of two highly-conserved cysteine amino acids near the amino terminus of these proteins. Within the CXC and CC subgroups, chemokines are further grouped into related families based on amino acid sequence similarity between them. CXC chemokine families include the IP-10 and Mig family; the GROα, GROβ, and GROγ family; the interleukin-8 (IL-8) family; and the platelet factor 4 (PF4) family; other CXC chemokines that have been identified are: C10, DC-CK1, CKα1, CKα2, ENA-78, GCP-2, and platelet basic protein (PBP) and its derivatives CTAPIII, β-thromboglobulin, and NAP-2. CC chemokine families include the monocyte chemoattractant protein (MCP) family including MCP-1 to MCP-4; the family including macrophage inhibitory protein-1α (MIP-1α), macrophage inhibitory protein-1β (MIP-1β), and regulated on activation normal T cell expressed (RANTES) protein; and the lymphotactin family; other CC chemokines that have been identified are: ATAC, eotaxin, eotaxin2, I-309, HCC-1, HCC-2, HCC-3, LARC/MIP-3α, MIP-3β, PARC, TARC, 6Ckine, ELC, SLC, CKβ4, CKβ6, CKβ7, CKβ8, CKβ9, CKβ11, CKβ12, CKβ13. CX3C (or CX3C) is a recently identified member of a new class of chemokines. The chemokines stromal cell-derived factor 1α (SDF-1α) and stromal cell-derived factor 1β (SDF-1β) form a chemokine family that is approximately equally related by amino acid sequence similarity to the CXC and CC chemokine subgroups. Individual members of the chemokine families are known to be bound by at least one chemokine receptor, with CXC chemokines generally bound by members of the CXCR class of receptors (CXCR1 -CXCR4), and CC chemokines by members of the CCR class of receptors (CCR1 -CCR8). For example, SDF-1α is known to be a ligand for the CXCR receptor fusin/CXCR4, and MIP-1α is bound by the CCR receptors CCR1, CCR4, and CCR5. Other chemokine receptors that have been identified are: BLR1, MDR15, EBI-1, CMKBRL1, HCMV-US28, HSV-ECRF3, and Duffy antigen (DARC).
The presence of a chemokine gradient attracts migratory cells such as lymphocytes, leukocytes, and antigen-presenting cells (APCs) that may participate in autoimmune reactions, inflammation, or normal immune responses, or that may release other intercellular factors to stimulate or inhibit angiogenesis, bone resorption, or other cellular processes. For example, the initiation of autoimmune disease requires the infiltration or recruitment of lymphocytes able to respond against self proteins into the organ bearing the antigenic self proteins. Inflammatory atherosclerotic lesions are due in part to infiltration of the vascular compartment by leukocytes recruited to the site. To induce an immune response, antigenic proteins and glycoproteins must bind to the surface of B lymphocytes to stimulate antibody production, and must be taken up by antigen-presenting cells, processed, and represented to T lymphocytes to mediate a T-lymphocyte response. Migratory cells that secrete IP-10 or IL-8, when attracted by a chemokine gradient to a particular site, respectively may inhibit or stimulate the formation of blood vessels at that site. Chemokines may be used to establish a chemoattractive gradient for migratory cells that are expressing the appropriate chemokine receptors, or to obscure an existing chemoattractive gradient.
Chemokine receptors are also involved in functions other than chemotaxis, such as interacting with viral proteins. HIV-1 is known to bind to certain proteins on the surface of cells in order to gain entrance into these cells and replicate or integrate the viral gene into the host DNA. The CD4 protein on T lymphocytes and other cells, including certain antigen presenting cells, has been shown to be bound by the HIV-1 viral envelope protein gp120. This is believed to induce in gp120 a conformational change that then exposes regions of gp120 and perhaps CD4 that subsequently bind to a chemokine receptor. To date CXCR4 (also known as fusin), CCR5, and several other chemokine receptors have been identified as co-receptors for HIV-1. Monocyte-tropic (M-tropic) isolates of HIV-1 require interaction with CCR5 in order to infect cells, while T-lymphocyte-tropic (T-tropic) HIV-1 isolates require another coreceptor, CXCR4, for infection. There is some evidence indicating that HIV-1 can also use other CCR receptors such as CCR2 and CCR3 to gain entry into cells. For some HIV-2 isolates, it appears that certain chemokine receptors such as fusin/CXCR4 alone can provide the cell-surface protein needed for binding and entrance into the cell.
There is a continuing requirement for new compositions that will enhance, alter, or inhibit chemokine-receptor interactions, and for methods for their use.